TIGAR overexpression diminishes radiosensitivity of parotid gland fibroblast cells and inhibits IR-induced cell autophagy.
ABSTRACT: Our previous study proved that TP53-induced glycolysis and apoptosis regulator (TIGAR) abrogation is able to radiosensitize glioma cells. Whether TIGAR over-expression has radio-protective effect in human parotid gland cells is still unknown. In this study human parotid gland fibroblast Hs 917.T cells were transfected with pcDNA3.1-TIGAR, and clonogenic assay was performed to investigate the radiosensitivity of Hs 917.T cells over-expressing pcDNA3.1 or pcDNA3.1-TIGAR. Western blot was carried out to demonstrate the autophagy activity of cells being irradiated, and immunofluorescence assay was used to evaluate the DNA damage repair process of irradiated Hs 917.T cells. It was revealed that TIGAR over-expression could diminish the radiosensitivity of Hs 917.T cells, and the autophagy level induced by ionizing radiation (IR) was also decreased by TIGAR transfection. The mechanism might rely on TIGAR over-expression induced ROS scavenging and NADPH increasing. Using autophagy inhibitor, it was also elaborated that IR-induced autophagy in Hs 917.T cells was protective autophagy but not traumatic autophagy.
Project description:<h4>Objective</h4>Hyposalivation-related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro.<h4>Materials and methods</h4>Single cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self-renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response.<h4>Results</h4>Single cells derived from parotid gland organoids were able to extensively self-renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid-derived stem cells displayed radiation dose-response curves similar to the submandibular gland.<h4>Conclusions</h4>Murine parotid gland organoids harbor stem cells with long-term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.
Project description:The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours.
Project description:In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.
Project description:Radiotherapy using high linear energy transfer (LET) radiation results in effectively killing tumor cells while minimizing dose (biological effective) to normal tissues to block toxicity. It is well known that high LET radiation leads to lower cell survival per absorbed dose than low LET radiation. High-linear energy transfer (LET) neutron treatment induces autophagy in tumor cells, but its precise mechanisms in osteosarcoma are unknown. Here, we investigated this mechanism and the underlying signaling pathways. Autophagy induction was examined in gamma-ray-treated KHOS/NP and MG63 osteosarcoma cells along with exposure to high-LET neutrons. The relationship between radiosensitivity and autophagy was assessed by plotting the cell surviving fractions against autophagy levels. Neutron treatment increased autophagy rates in irradiated KHOS/NP and MG63 cells; neutrons with high-LETs showed more effective inhibition than those with lower LET gamma-rays. To determine whether the unfolded protein response and Akt-mTOR pathways triggered autophagy, phosphorylated eIF2? and JNK levels, and phospho-Akt, phosphor-mTOR, and phospho-p70S6 levels were, respectively, investigated. High-LET neutron exposure inhibited Akt phosphorylation and increased Beclin 1 expression during the unfolded protein response, thereby enhancing autophagy. The therapeutic efficacy of high-LET neutron radiation was also assessed in vivo using an orthotopic mouse model. Neutron-irradiated mice showed reduced tumor growth without toxicity relative to gamma-ray-treated mice. The effect of high-LET neutron exposure on the expression of signaling proteins LC3, p-elF2a, and p-JNK was investigated by immunohistochemistry. Tumors in high-LET-neutron radiation-treated mice showed higher apoptosis rates, and neutron exposure significantly elevated LC3 expression, and increased p-elF2a and p-JNK expression levels. Overall, these results demonstrate that autophagy is important in radiosensitivity, cell survival, and cellular resistance against high-LET neutron radiation. This correlation between cellular radiosensitivity and autophagy may be used to predict radiosensitivity in osteosarcoma.
Project description:Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found recently in mouse models that transient activation of Hedgehog pathway following irradiation rescued salivary gland function by preserving salivary stem/progenitor cells, parasympathetic innervation and microvessels. Due to huge differences between salivary glands of rodents and humans, to examine the translational potential of this approach, we evaluated effects of Shh gene transfer in a miniature pig model of irradiation-induced hyposalivation. <b>Methods</b>: The right parotid of each pig was irradiated with a single dose of 20 Gray. Shh and control GFP genes were delivered into irradiated parotid glands by noninvasive retrograde ductal instillation of corresponding adenoviral vectors 4 or 16 weeks after irradiation. Parotid saliva was collected every two weeks. Parotid glands were collected 5 or 20 weeks after irradiation for histology, Western blot and qRT-PCR assays. <b>Results</b>: Shh gene delivery 4 weeks after irradiation significantly improved stimulated saliva secretion and local blood supply up to 20 weeks, preserved saliva-producing acinar cells, parasympathetic innervation and microvessels as found in mouse models, and also activated autophagy and inhibited fibrogenesis in irradiated glands. <b>Conclusion</b>: These data indicate the translational potential of transient activation of Hedgehog pathway to preserve salivary function following irradiation.
Project description:Highlights • RBPMS-AS1 and CAMTA1 are lowly expressed in GBM patients.• RBPMS-AS1 and CAMTA1 enhance the radiosensitivity of GBM.• miR-301a-3p diminishes the radiosensitivity of GBM.• RBPMS-AS1 enhances CAMTA1 expression in GBM cells through sponging miR-301a-3p.• CAMTA1 promotes NRGN transcription. <h4>Objective</h4> Glioblastoma (GBM) is the most frequent brain malignancy with high incidence, and long noncoding RNAs (lncRNAs) exerts functions in GBM. In this research, we focused on the capabilities of lncRNA RBPMS-AS1 in radiosensitivity of GBM. <h4>Methods</h4> RBPMS-AS1 and CAMTA1 expression levels were determined in GBM tissues and cells. StarBase v3.0 database was searched for predicting miRNAs that simultaneously bound to RBPMS-AS1 and CAMTA1. pcDNA3.1-RBPMS-AS1, pcDNA3.1-CAMTA1, miR-301a-3p mimic, or pcDNA3.1-RBPMS-AS1/pcDNA3.1-CAMTA1 and miR-301a-3p mimic were transfected into GBM cells to test radiosensitivity, cell proliferation and apoptosis. The interactions of miR-301a-3p with RBPMS-AS1 and CAMTA1, as well as CAMTA1 and NRGN, were confirmed. In vivo imaging technology was utilized to detect tumor growth in orthotopic xenograft tumors, and Ki67 expression was tested in intracranial tumors. <h4>Results</h4> RBPMS-AS1 and CAMTA1 levels were reduced in GBM tissues and cells. miR-301a-3p had a binding site with both RBPMS-AS1 and CAMTA1 and it was the most significantly-upregulated one. Upregulation of RBPMS-AS1 or CAMTA1 enhanced the radiosensitivity and cell apoptosis while suppressing proliferation of GBM cells. Conversely, miR-301a-3p overexpression diminished the radiosensitivity and cell apoptosis while inducing proliferation of GBM cells. Overexpression of RBPMS-AS1 or CAMTA1 reversed the effects of overexpressed miR-301a-3p in GBM cells. Mechanistically, RBPMS-AS1 enhanced CAMTA1 expression in GBM cells through sponging miR-301a-3p, and CAMTA1 promoted NRGN expression. In animal experiments, overexpressed RBPMS-AS1 inhibited tumor growth and the positive expression of Ki67 both before and after radiation therapy. <h4>Conclusion</h4> RBPMS-AS1 promotes NRGN transcription through the miR-301a-3p/CAMTA1 axis and enhances the radiosensitivity of GBM.
Project description:Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to hypomethylation, DAC in AML is also involved in cell metabolism, apoptosis, and immunity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) functions to inhabit glycolysis and protect cancer cells from reactive oxygen species- (ROS-) associated apoptosis. Our previous study revealed that TIGAR is highly expressed in myeloid leukemia cell lines and AML primary cells and associated with poor prognosis in adult patients with cytogenetically normal AML. In the present study, it was found that in a time- and concentration-dependent manner, DAC downregulates the TIGAR expression, induces ROS production, and promotes apoptosis in HL-60 and K562 cells. However, blocking the glycolytic pathway partially reversed the combined effects of DAC and TIGAR knockdown on apoptosis, ROS production, and cell cycle arrest, indicating that DAC induced apoptosis through the glycolytic pathway. Furthermore, TIGAR also has a negative impact on autophagy, while DAC treatment upregulates autophagy-related proteins LC3, Beclin-1, ATG3, and ATG-5, downregulates p62, and promotes the formation of autophagosomes, indicating that DAC may activate autophagy by downregulating TIGAR. Taken together, DAC plays an unmethylated role in inducing apoptosis and activating autophagy in myeloid leukemia by downregulating TIGAR.
Project description:Heavy-ion radiotherapy has a potential advantage over conventional radiotherapy due to improved dose distribution and a higher biological effectiveness in cancer therapy. However, there is a little information currently available on the cellular and molecular basis for heavy-ion irradiation-induced cell death. Autophagy, as a novel important target to improve anticancer therapy, has recently attracted considerable attention. In this study, the effect of autophagy induced by high linear energy transfer (LET) carbon ions was examined in various tumor cell lines. To our knowledge, our study is the first to reveal that high-LET carbon ions could induce autophagy in various tumor cells effectively, and the autophagic level in the irradiated cells increased in a dose- and LET-dependent manner. The ability of carbon ions to inhibit the activation of the PI3K/Akt pathway rose with increasing their LET. Moreover, modulation of autophagy in tumor cells could modify their sensitivity to high-LET radiation, and inhibiting autophagy accelerated apoptotic cell death, resulting in an increase in radiosensitivity. Our data imply that targeting autophagy might enhance the effectiveness of heavy-ion radiotherapy.
Project description:The p53-inducible TIGAR protein functions as a fructose-2,6-bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy--a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.
Project description:Hyperglycemia-induced neuronal apoptosis is one of the important reasons for diabetic neuropathy. Long-time exposure to high glucose accelerates many aberrant glucose metabolic pathways and eventually leads to neuronal injury. However, the underlying mechanisms of metabolic alterations remain unknown. TP53-inducible glycolysis and apoptosis regulator (TIGAR) is an endogenous inhibitor of glycolysis and increases the flux of pentose phosphate pathway (PPP) by regulating glucose 6-phosphate dehydrogenase (G6PD). TIGAR is highly expressed in neurons, but its role in hyperglycemia-induced neuronal injury is still unclear. In this study, we observed that TIGAR and G6PD are decreased in the hippocampus of streptozotocin (STZ)-induced diabetic mice. Correspondingly, in cultured primary neurons and Neuro-2a cell line, stimulation with high glucose induced significant neuronal apoptosis and down-regulation of TIGAR expression. Overexpression of TIGAR reduced the number of TUNEL-positive neurons and prevented the activation of Caspase-3 in cultured neurons. Furthermore, enhancing the expression of TIGAR rescued high glucose-induced autophagy impairment and the decrease of G6PD. Nitric oxide synthase 1 (NOS1), a negative regulator of autophagy, is also inhibited by overexpression of TIGAR. Inhibition of autophagy abolished the protective effect of TIGAR in neuronal apoptosis in Neuro-2a. Importantly, overexpression of TIGAR in the hippocampus ameliorated STZ-induced cognitive impairment in mice. Therefore, our data demonstrated that TIGAR may have an anti-apoptosis effect <i>via</i> up-regulation of autophagy in diabetic neuropathy.